This is a revised competing renewal application for a RO-1 in its 12th year, with the overarching goal of investigating the function of the CD14-TLR4 pathway in the liver. The liver is a central regulator of the systemic immune response following trauma, warm ischemia/reperfusion (I/R), or sepsis, and is the primary site for clearance of bacterial endotoxin (lipopolysaccharide: LPS). The CD14-TLR4 receptor system is now known to mediate innate immune responses to both microbial invasion and sterile tissue injury. We hypothesize that this innate immune recognition system in the liver serves at least two functions. First, CD14-TLR4 permits recognition of danger, whether from pathogens or damaged tissue. Second, the system is utilized to rapidly clear microbial products such as LPS. Both functions require components of the CD14- TLR4 system and both lead to downstream signaling events that impact on cell type-specific functions. We will address these issues in two interrelated aims. In Aim 1, our overarching goal is to determine the mechanisms for LPS clearance by the CD14-TLR4 pathway in the liver. In Aim 2, we propose to determine the mechanisms of TLR4-mediated inflammation in hepatic warm I/R. For both Aims, we will utilize mice to take advantage of strains defective in components of TLR4 function and signaling. To study specific aspects of cell function and signaling, we will carry out experiments using primary (hepatocyte [HC[ and nonparenchymal [NPC]) cell culture as we have for nearly two decades. Where feasible, confirmation of the in vitro observations will be performed in vivo. To discriminate between the contribution of NPC and HC in vivo, we will use chimeric mice as we have done in the past. Our design will be specific to the hypothesis. For Aim 1, we will study only LPS uptake and processing. For Aim 2, warm I/R will be used as the model system of local tissue injury. Insights gained from these studies should significantly enhance our evolving understanding of the common responses of the liver to sudden disruptions of homeostasis, both from pathogens and from damaged tissue.